Expression of MammalianMetallothionein-I Gene inCyanobacteria to Enhance Heavy MetalResistance1

Z. CHEN , L. REN , Q. SHAO , D. SHIà and B. RU * College of Life Science, Peking University, Beijing, People's Republic of ChinaàInstitute of Botany, Chinese Academy of Science, Beijing, People's Republic of China

Metallothionein (MT) from mammals is a low molecularweight (about 6±7 KD), and cystein-rich (20 cystein permolecular) protein (Vallee, 1991). So MT has the strongbinding ability of metal such as Cd, Hg and Pd. Wild-typed cyanobacteria have their own metallothionein-likeproteins, which are of low cysteine content and have weakability to bind heavy metal. Therefore, it is imperative totransfer the MT-I gene from mammal into cyanobacteriacell for the absorption and accumulation of heavy metalsin aqueous system including marine and lake (Misra andGedamu, 1989). We inserted the MT-I cDNA gene afterthe strong promoter PpsbA or Psmt into the intermediaryvector pRL-439, and then pRL-MT was ligated withshuttle vector pKT-210 (a shuttle vector pDC-08 was usedat an earlier time) to construct the shuttle expressionvector pKT-MT. (The MT-I mutant a-KKS-a also wasselected as aim gene.) Then the constructed vector wasintroduced into cyanobacterium Anabaena sp. PCC7120,Synechocystis sp. PCC6803 and Synechococcus sp.PCC7942 with triparental conjunctive transfer (Bryant,1994). The expression e�ciency is about 1%. We alsoused homologous recombination vector pTZ18-8 to in-troduce MT-I cDNA into chromosomes in cyanobacteriaand got higher expression e�ciency. Metal tolerance testsshow that the transgenic cyanobacteria acquire metal re-sistance up to 60 lmol/g CdCl2 and expression e�ciencyup to 1045 lg MT/g fresh cells according to the data ofELISA. Ó 1999 Published by Elsevier Science Ltd. Allrights reserved.

Keywords: metallothionein; metallothionein mutant gene;transgenic cyanobacterium; aqueous pollution; heavymetal tolerance; triparental conjugate transfer; homolo-gous recombination.

Introduction

Today water pollution is becoming an increasinglyserious problem with the development of modern in-dustry and agriculture. The drainage of sewage resultsin contamination of aqueous system including marine,lake and river. Among the heavy metals that pollutedthe aqueous system, cadmium (Cd) is most serious,then mercury (Hg) and lead (Pb). Heavy metals, suchas cadmium can move into the food chain and dogreat harm to animal, even ®nally to human health.So it is imperative to solve the problem, and the ap-plication of heavy metal ion-resistant microbes inpolluted e�uents has become a focus for bioremedia-tion.

Metallothionein is a low molecular weight, cysteine-rich, metal binding protein owned by a variety of speciesincluding mammals, plants and microorganisms.Mammalian MT contains 20 cysteine residues out of61 amino acid residues, and fold into two separate do-mains: one with 11 cysteine residues and binding withfour X2� ions, another with the other cystein residuesand three X2� ions. The a-domain has power ability tobind Cd2� and Hg2� ions, and the b-domain is easy tobind with Zn2�ion. Each domain binds metal ions in apolynuclear metal-thiolate cluster with ligation throughthiolates of the cysteine residues, so the ligation is verystrong. Some experiments show that each domain canfunction dependently. These characteristics make MTplay an important role in heavy metal detoxi®cation andmetal ion de®ciency disease.

Cyanobacteria are photosynthetic prokaryotesadapted to the most ecological environments Marsacand Gedamu (1975). As the easiness of natural or arti-®cial cultivation with low coat and available vectorsystem, cyanobacteria may be the best host for the ex-pression vector inserted with MT gene. Transgenic MTcyanobacteria can be used as a ®lter for removal ofheavy metals from contaminated waters.

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Ó 1999 Published by Elsevier Science Ltd. All rights reserved

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*Corresponding author.1 Supported by National Ninth Five Year, Key Technology Program

on Biotechnology in China 96-C02-04-05.

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Methods and Results

Use pKT-210 to construct shuttle expression vector pKT-MT and get MT transgenic cyanobacterium with themethod of triparental conjugate transfer

Plasmid pKT-210 has both Sm and Cm resistance.The shuttle expression vector pKT-MT has additionalAmp resistance because of insertion of pRL-439. Plas-mid pKT-210 also contains fragment RSF1010, which isin charge of duplication of the plasmid. Transgenic cy-anobacterium including vector pKT-MT can be moreeasily selected and get higher expression e�ciency thanthat was transfered with vector pDC-MT in our earlierexperiments. Usually our selected host stains includeAnabaena sp. PCC7120, Synechocystis sp. PCC6803 andSynechococcus sp. PCC7942.

A strong promoter PpsbA was used for high e�ciencyexpression

The promoter psbA comes from chloroplast DNA ofAmaranthus hybrids. PpsbA was a strong promoter inboth plant and cyanobacterium, also in E. coli. Only ifcyanobacteria is growing normally, PpsbA takes its role.The construction of pKT-psbA-MT is showed in Fig. 1.The MT expression e�ciency was about 600 lg/g freshcells, 15 times to 40 lg/g fresh cells in transgenic cy-anobacterium with vector pDC-MT.

The promoter smt (O-P) was a better promoter forincreasing the expression e�ciency

The smt (O-P) from wild-typed cyanobacteria was astrong promoter induced by metal ion. So it is appro-priate for us to use this promoter to promote the ex-pression of foreign gene MT-IA cDNA in order toenhance the heavy resistance of transgenic cyanobacte-ria. The shuttle expression plasmid pKT-MAC-MT wasconstructed from pBX-MT plasmid, pRL-439 plasmidand pKT-210 plasmid and promoter smt (O-P) (alsocalled MAC). Except for the di�erence of promoters,

pKT-MAC-MT is the same with pKT-psbA-MT.Southern blotting Davis et al. (1986) and western blot-ting indicate the success of cloning and expression (700lg/g fresh cells). A metal tolerance experiment showedthat the transgenic cyanobacterium acquired metal re-sistence to 50 lmol/l CdCl2:

Double promoter was further used to increase theexpression e�ciency of mammal MT gene

As both the psbA and MAC are strong promoters incyanobacteria, we use double promoters to promote theexpression of the MT gene. The construction of shuttleexpression plasmid pKT-psbA-MAC-MT was similar tothat of pKT-MAC-MT, the only di�erence was thatdouble promoter psbA and smt were used together in-stead of a single promoter to promote the expression ofMT-I gene. As a result, the transgenic cyanobacteriatrain has acquired an increased resistance to 60 lmol/lCdCl2. The expression e�ciency was also increased to be800 lg/g fresh cells. The above three transgenic cyano-bacteria were all determined byWestern Blotting (Fig. 3).

Insert MT gene mutant a-KKS-a and b5 into shuttleexpression vector for the remedy of de®ciency disease andclearance of water pollution

As cadmium (Cd) pollution is most serious among allheavy metal pollution in nature, we intend to construct aspecial transgenic cyanobacteria with special strongability to bind and remove the Cd pollution in the watersystem. As we know, the a-Domain of metallothioneinhas a far stronger inclination to bind Cd than b-domainNielson and Winge (1984), therefore, the a-domain cantake the place of b-domain in MT and conform an a-KKS-a mutant. The a-KKS-a mutant will have morespecial ability to accumulate metal Cd. We veri®ed thatmutant gene has a powerful function: the cyanobacteriawith pKT-a-KKS-a acquired the highest cadmium re-sistance up to 70 lmol/l. The experiments of oxygen-evolving activity indicate that the expressed mammal

Fig. 1 Construction of the shuttle expression plasmid pKT-MT.

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MT protected the transgenic cyanobacterium from be-ing destroyed by cadmium Marsac and Houmard (1987)(Fig. 4).

Use of pTZ-18-8 plasmid to construct shuttle expressionvector pTZ-MT, and then get MT transgenic cyanobac-terium with the method of homologous recombination

A downstream fragment (about 1870 bp) of the psbBgene of Synechocystis sp. PCC 6803 that acted as anintegration platform was ampli®ed by PCR and insertedinto Phagmid pTZ18U to construct the integrationvector pTZ18-8. Then the Human liver Metallothionein(hMT) gene following the promoter PpsbA was cut fromPlasmid pRL-MTand inserted into the integration vec-tor pTZ18-8 to construct the integration vector pTZ-

MT, which was introduced into Synechocystis sp. PCC6803 by natural transformation. A homologous recom-bination takes place between the psbB fragment (about1807 bp) in the pTZ-MT and psbB gene sequence in thechromosome of transformat. As a result, the hMT genewas integrated into the chromosome of the transgenicSynechocystis sp. PCC 6803 as shown above in Fig. 2.The experiment of continuous culture tells us thattransgenic cyanobacterium with Amp resistance andmetal resistance has been obtained. The Southern blot-ting showed the presence of the hMT gene in the chro-mosome of transgenic cyanobacterium. The Westernblotting demonstrated the expression of the integratedhMT gene. A further metal tolerance test showed thatthe transgenic cyanobacterium acquired metal resistance

Fig. 2 Construction of the integration vector pTZ-MT.

Fig. 3 Con®rmation of the expression of the MT-IA cDNA in trans-genic Synechocystis sp. PCC 6803 by Western blot. Lane A:sample from transformant (with psbA promoter), Lane B:sample from transformant (with MAC promoter), Lane C:sample from transformant (with psbA-MAC promoter), LaneD: wild cyanobacteria, Lane E: standard MT.

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to be 60 lmol/l CdCl2, 6 times higher than that of wildtype cells. From the data of ELISA, the expression ef-®ciency was determined to be 1045 lg MT/g fresh cells(Fig. 5).

Discussion

We have constructed a series of transgenic cyano-bacteria strains with more and more high heavy metaltolerance. We think that both triparental conjugatetransfer and homologous recombination are e�cient,and each has its unique merit. Therefore, we want pKT-210 to make more copies in its host and pTZ-18 highertransfer e�ciency. Our experiments show that MT mu-tant will be more ideal than native MT, so some furtherresearch work on MT gene such as construction of an

has been carried on. The fact that transgenic cyano-bacteria with double promoter can get higher expression

can give us some inspiration. The next step we need todo is to make the strains available in practice. The so-lidi®cation of cyanobacteria will be on our way.

This work received ®nancial support from National Ninth Five year,Key Technology Program on Biotechnology in China 96-C02-04-05.The authors would like to thank Prof. C. P. Wolk of Michigan StateUniversity for providing plasmids pRL 439 and pKT210.

Vallee, B. L. (1975) Introduction on Metallothionein. MethodsEnzymol. 205, 3±7.

Misra, S. and Gedamu, L. (1989) Theoretical and Applied Genetics 78,161±168.

Bryant, D. A. (1994) The Molecular Biology of Cyanobacteria, pp. 583±588.

Marsac, N. J. and Gedamu, L. (1975) Adaptation of cyanobacteria tonenvironment stimuli. FEMS Microbiological Review 104, 119±190.

Davis, L. G., Diber, M. D. and Battery, J. F. (1986) Basic Methods inMolecular Biology, Elsevier, Paris, pp. 305±309.

Nielson, K. B. and Winge, D. (1984) Journal of Biological Chemistry259, 4942±4946.

Marsac, N. J. and Houmard, J. (1987) Advances in CyanobacterialMolecularGenetics.The Cyanobacteria, Elsevier, Paris, pp. 251±302.

Fig. 5 Content of MT in di�erent transgenic cyanobacteria.

Fig. 4 The comparison between the oxygen-evolving activities of wildtype and transgenic Synechocystis sp. PCC 6803 in the BG-11medium supplemented with 10 lmol/l CdCl2 (r): Transgenic(j): Wild type.

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